La Subjetividad Política en el marco de la realidad Juvenil

Figure 2.13: Schematic Representation of GroEL/GroES Assisted Folding: Unfolded substrate polypeptide binds to the trans ring of GroEL. ATP-dependent domain movement of the apical GroEL domains result in stretching of tightly bound regions of the substrate and in release and partial compaction of less stably bound regions of substrate. GroES binds after ATP binding leading to substrate encapsulation. Folding occurs in the chaperonin cage in the time scale ~10 s regulated by ATP hydrolysis rate. Upon binding of GroES and ATP in the other ring, GroES dissociates leading to substrate release. Incompletely folded substrate again binds to GroEL for the next folding attempt. Adapted from (Hartl and Hayer-Hartl, 2009).

The reaction cycle of GroEL/ES system have been investigated extensively (Hartl and Hayer-Hartl, 2009; Horwich et al., 2009). Under physiological conditions, GroEL is in the bullet form, the acceptor state of the GroEL for the protein (Rye et al., 1999). Cis ring of GroEL bullet has GroES and ADP bound and the trans-ring is in the substrate acceptor state. Unfolded polypeptide and ATP binds to the trans-ring of GroEL which triggers specific conformational changes resulting in the binding of GroES and subsequent release of bound polypeptide in the cis hydrophilic cavity. An encapsulated polypeptide of ~50 kDa is free to fold in this environment for 10-15 s at 25°C, the time needed for cooperative ATP hydrolysis. Subsequent ATP and GroES binding to the opposite GroEL ring causes the dissociation of GroES and release of substrate. Non-native protein is rapidly recaptured by GroEL for another folding attempt (Figure 2.13). Proteins that exceed the size limit of the chaperonin cage either use the Hsp70 system for folding (Agashe et al., 2004; Kerner et al., 2005) or may reach their

native state through binding and release from GroEL without encapsulation (trans-folding) (Chaudhuri et al., 2001), which is still an open question.

Several possible mechanisms have been proposed to explain the rate acceleration of folding of substrate proteins in presence of GroEL/ES system. Broadly, these mechanisms may be classified as active or passive. In a passive folding mechanism, GroEL is thought to have no direct effect on the conformation of a non-native protein, serving simply to block inhibitory aggregation and free energy of ATP hydrolysis is used only to assemble or disassemble an isolation chamber. An active model, by contrast, involves the direct modification of a substrate protein‟s accessible conformational space while folding. Still,

GroEL mechanism of protein folding is debated.

Passive binding of an aggregation-prone intermediate by GroEL can block aggregation, but captured proteins must eventually be released back into the free solution in order to complete the final steps of folding or oligomer assembly. Folding takes place at infinite dilution, with each GroEL-associated protein physically isolated from other aggregation-prone proteins (Ellis, 1994a, b; Saibil et al., 1993). A GroEL ring is imagined to shift between high and low affinity states for a non-native substrate protein which is driven by ATP hydrolysis and GroES binding (Badcoe et al., 1991; Martin et al., 1991). This model of GroEL- mediated protein folding is known as the Anfinsen cage model (Ellis, 1994b; Saibil et al., 1993). Essentially, the enclosed cavity is envisioned to be near ideal, isolated environment where folding could proceed unhindered, propelled only by the intrinsic thermodynamic drive encoded by the protein amino acid sequence. But the purely passive model ignores the possibility that dominant kinetic traps could be misfolded states that have little or no possibility of accessing the native state.

One efficient way to bypass local energy minima is unfolding of substrate protein when bound to GroEL. Two general mechanisms, one thermodynamic and one catalytic, have been suggested to explain how GroEL could induce unfolding of substrate proteins. In the thermodynamic partitioning model (Zahn et al., 1994a; Zahn et al., 1994b), GroEL preferentially binds less folded conformations within an ensemble of non-native states and thereby shifts a pre-existing, intrinsic equilibrium towards less folded states without unfolding. In contrast, catalytic unfolding model suggests that GroEL could catalytically drive unfolding by lowering the free energy barriers that separate different folded states from one another. However, a direct connection between stimulated folding and the disruption of intra-molecular structure in a non native and stringent protein has been difficult to establish.

An alternate mechanism for unfolding was proposed by Lorimer and colleagues who suggested that substrate protein unfolding could be directly linked to the ATP-driven structural rearrangements of the GroEL ring itself (Lin et al., 2008; Shtilerman, 1999) which was termed as „Forced Unfolding model‟ or „Itearative Annealing model‟. It suggests that the ATP-driven elevation and rotation of the GroEL apical domains that are necessary for GroES binding might apply a mechanical strain to a non-native protein bound across multiple GroEL apical domains. The forced protein unfolding in consecutive rounds of chaperonin binding promotes the reversal of kinetically trapped states. Consistent with such a mechanism, proteins undergo a conformational expansion on initial binding to GroEL and upon subsequent ATP binding (Lin et al., 2008; Sharma et al., 2008). A single ring mutant of GroEL, SR-EL, exists as a single, seven subunit ring that possess normal ATP binding, hydrolysis and GroES binding like GroEL (Weissman et al., 1995a; Weissman et al., 1996). Since SR-EL lacks the signal from the trans ring, it can only complete single round of binding and encapsulation of the substrate polypeptide. Remarkably, SR-EL is fully capable of driving productive folding of stringent substrate proteins (Brinker et al., 2001; Rye et al., 1997; Tang et al., 2006; Weissman

et al., 1996) without unfolding cycles. Also, many hydrogen-deuterium exchange experiments and FRET measurements failed to detect evidence for forced unfolding upon ATP and GroES binding to a substrate protein bound to SR-EL (Chen et al., 2001; Lin and Rye, 2004; Park et al., 2005). Also there is no evidence for the extensive and steady conformational progression predicted by continuous annealing.

On the other hand, the confinement model of protein folding suggests that the physical properties of the cavity are likely to have additional effects on folding beyond prevention of aggregation. Spatial restriction of a non-native protein may narrow a protein‟s folding funnel, physically excluding large regions of conformational space and confining the subsequent search for the native state to a smaller range of states. Effectively, both smoothening and narrowing the folding funnel, in theory, can result in accelerated folding. Recently, confinement of the substrate polypeptide has been shown to have direct influence on rate of protein folding (Brinker et al., 2001; Tang et al., 2006). Mutational analysis has been done to show that the polar residues of the cavity wall are crucial for rapid folding (Brinker et al., 2001; Lin and Rye, 2004; Tang et al., 2006). According to molecular dynamics simulations, these polar residues are expected to promote folding by accumulating ordered water molecules in their vicinity, thereby generating a local environment in which a substrate polypeptide is forced to bury exposed hydrophobic residues more effectively (England et al., 2008). Dynamic interaction between non-native folding intermediates and a weakly hydrophobic cavity wall has been suggested to accelerate folding by lowering the free energy barriers between different states (Betancourt and Thirumalai, 1999; Chan and Dill, 1996). This enhancement could be due to stabilization of more hydrophobic and less folded conformational transition states, providing an additional smoothening of the free energy landscape. Alternately, the GroEL/ES cavity has been suggested to increase ruggedness of a broad and slowly crossed transition state

ensemble, opening folding routes that are not significantly populated in bulk solution (Jewett et al., 2004).